Fig 1.
Phylogenetic relationships and chromosome characteristics of F. fujikuroi isolates and related species.
(A) World map modified after https://commons.wikimedia.org/wiki/File:BlankMap-World6.svg. Colored dots present the origin of the different F. fujikuroi isolates (see Table 1). (B) Maximum likelihood tree showing phylogenetic relationships of F. fujikuroi isolates and other species representing the Asian, African and American clades of the Fusarium fujikuroi complex (FFC), as well as F. oxysporum, and the distantly related species F. langsethiae and F. avenaceum. It was calculated based on the protein sequences of 5,181 single copy genes that are shared among all analyzed species. (C) and (D) Separation of chromosomes of the nine F. fujikuroi isolates and F. oxysporum V-64-1 as outgroup on a CHEF gel. Chromosomes of Schizzosaccharomyces pombe and Saccharomyces cerevisiae were used as size standards.
Table 1.
Strains sequenced in this study or used for comparison.
Table 2.
Comparative genome statistics for all sequenced F. fujikuroi isolates and F. oxysporum V64-1 (outgroup).
Table 3.
Distribution of selected gene families in genome sequences of Fusarium fujikuroi isolates and F. oxysporum V64-1 (outgroup).
Fig 2.
Phenotypic characteristics of the nine F. fujikuroi isolates and F. oxysporum V64-1 as outgroup.
Colony morphology of the strains grown on solidified complete medium from the top (A) and the bottom (B) of the plates. Variation in pigmentation of the strains grown in liquid synthetic medium containing 6 mM glutamine (C) (optimal for bikaverin), 60 mM glutamine (D) or 6 mM NaNO3 (E) (optimal for fusarubins). (F) Microscopic images of microconidia and macroconidia.
Fig 3.
Content and arrangement of genes in different gene clusters with variations in the single isolates.
The PKS51 (A) and PKS40 (B) gene clusters are present only in strain B14. (C) The PKS13, PKS17, PKS18, and PKS8 gene clusters at the end of chromosome 11 are missing in strain NCIM 1100. Arrows in blue represent genes belonging to a specific gene cluster. Green arrows represent genes that have closely related homologs in two or more isolates while light-gray arrows represent genes that do not have closely related homologs in other isolates. Ψ indicates a pseudogene.
Fig 4.
Content and arrangement of genes in different gene clusters with variations in the single isolates.
(A) The PKS5 cluster seems to be functional only in strain B14. The PKS5-encoding gene is truncated or missing in the other isolates. (B) The PKS19 (fujikurin) gene cluster is present in some and totally missing in other isolates. (C) PKS16 at the end of chromosome 11 in F. fujikuroi IMI 58289 is truncated in stains C1995 and E282, and missing in strain NCIM 1100. Arrows in blue represent genes belonging to a specific gene cluster. Green arrows represent genes that have closely related homologs in two or more isolates while light-gray arrows represent genes that do not have closely related homologs in other isolates. Ψ indicates a pseudogene.
Table 4.
Secondary metabolite production of the ten analyzed strains in submerged culture at three different nitrogen and pH conditions.
Table 5.
Expression of the secondary metabolite key enzyme-encoding genes under high (A) and low (B) nitrogen conditions and in rice roots after 7 days post inoculation (dpi).
n.p.–cluster not present.
Fig 5.
Gibberellic acid (GA) and fumonisin production and gene expression in the ten analyzed strains.
A) GA (CPS/KS) and fumonisin (FUM2) gene expression studies by Northern blot analysis after 3 days of growth in synthetic medium with 6 mM glutamine. GA (B) and fumonisin (C) production levels after 7 days of growth in synthetic medium with 6 mM glutamine.
Fig 6.
Pathogenicity assay of the F. fujikuroi and F. oxysporum isolates.
(A) Symptoms in the rice stems that were inoculated with the fungal isolates and water only (control). The uppermost stem level of the rice seedling with control treatment is indicated by a thin dotted bar. (B) Symptoms in the rice roots after pathogen infection. (C) The plant heights and internode lengths of pathogen-infected rice seedling at 6 dpi. Error bars show standard deviations. The same letter above bars indicates no significant difference. n.d., not detected.
Table 6.
Analyses of secondary metabolites in rice after seven dpi.
Fig 7.
Shoot and root growth of rice seedlings 5, 7, and 9 days post inoculation (dpi) of F. fujikuroi B14 and gene deletion strains dervied from B14.
(A-C) Shoot growth. (D) Root growth. (A) Shoot and root growth 5 dpi with or without exogenous supply of fumonisin FB1 (1 μM), fusaric acid (FSA; 10 μM) or GA3 (10 μM). (B) Shoot growth 7 dpi. (C) Shoot growth 9 dpi. Mock: neither fungal inoculation nor toxin treatement; B14 (stunting pathotype) and B20 (bakanae pathotype) wild-type strains; Δfub1 (PKS6, fusaric acid) deletion strain; Δfum1 (PKS11, fumonisins) deletion strain; Δfub1/Δfum1 double deletion strain; ΔPKS51 (PKS51, unknown product).
Fig 8.
Population analysis of field isolates in Korea.
(A) Rice seedlings 9 dpi of F. fujikuroi field isolates obtained from Korea. Control: no fungal inoculation, 1: B14, 2: B20, 3: JA19, 4: JA35, 5: JA23, 6: JD8, 7: OS122, 8: B41, 9: B3, 10: B66, 11: V76, 12: V74, 13: 16-R18, 14: B27, 15: B17, 16: R19, 17: 16-R24. Pathogenicity test with 15 additional isolates revealed a clear separation between the bakanae and stunting pathotype. (B) A phylogenetic tree constructed by the NJ method using the nucleotide sequences of combined TEF1 and RPB2 from the two pathotypes of field isolates, determined by pathogenicity test (A), and from the worldwide isolates used in this study. The phylogenetic tree supports the separation of the two pathotypes into two different phylogenetic subclades.